Development of a novel method for the diagnosis of a disease based on genome information has been expected since the base sequence of the human genome was determined. For example, genes relating to various diseases such as cancers have been found from gene analyses based on the base sequence information of genomes. These disease-related genes and disease-related proteins are attracting attentions as diagnostic bio-markers for diseases such as cancers. However, under the current circumstance, a stable result cannot be obtained by a diagnosis by detecting a bio-marker, for the reasons that sufficient expression of the bio-marker cannot be necessarily obtained at an early stage of a disease, and the like.
Early diagnosis of diseases is essential for the future medical treatments from the viewpoints of improvement in cure rates and reduction of the burden by patients. As an approach for early diagnosis, a method comprising detecting the change in the promoter activity of a gene that expresses at an early stage of a disease. The probability of expression of the gene is controlled by the change in the activity of a gene promoter. The activity of gene promoters is observed earlier than the expression of disease-related genes and disease-related proteins.
As a method for detecting the activity of a gene promoter in a cell, a reporter gene assay may be exemplified. The reporter gene assay is a method comprising introducing a reporter vector in which an expression cassette to which a reporter gene for visualizing the activity of a promoter is bound is incorporated at the downstream of the promoter into a subject cell, and quantifying the activity of the promoter based on the activity of a reporter protein. As the reporter gene, a luciferase gene, a (β-galactosidase gene, a fluorescent protein gene and the like are adopted.
However, for example, at an early stage of a disease, the number of cell in a pre-disease state by the change in the activity of a gene promoter is quite little. It cannot be considered that the probability of incorporation of the cell in blood or a tissue collected for diagnosis is high. Therefore, a technique for detection with high sensitivity is required for early diagnosis of a disease and the like.
At present, as a reporter gene assay with high sensitivity, a method by a virus reporter vector in which a gene promoter and a fluorescent protein are incorporated in a genome of a virus has been reported. In this reporter gene assay, the sensitivity of the reporter gene assay is increased by increasing the amount of expression of the fluorescent protein by proliferating a virus vector in a host cell infected with the virus vector, thereby increasing the amount of expression of the fluorescent protein. However, a virus reporter vector utilizes the ability of infection of a virus for introducing the vector into a host cell. Therefore, there is a risk of infection of an operator with the virus reporter vector. Accordingly, the handling of the virus reporter vector is not convenient, and there is a problem in safeness.
On the other hand, a plasmid reporter vector is handled more conveniently than the virus reporter vector is, and has no infectiveness and has high safeness. Many reporter gene assays using a plasmid reporter vector have been reported. An application of a plasmid reporter vector to early diagnosis of diseases is expected. However, unlike a virus reporter vector, a plasmid vector does not proliferate in a host cell, and thus there is a problem in the detection sensitivity thereof.
The object of the embodiment is to provide a plasmid vector, a method for detecting a gene promoter and an assay kit, by which the activity of a gene promoter can be detected with high sensitivity.